Several publications are referenced in this application by numbers in parentheses in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.
Silent information regulator 2 (Sir2) proteins (also referred to as sirtuins) are well conserved across all kingdoms of life and are implicated in the control of gene silencing, apoptosis, metabolism, and aging (1-6). Sir2 is required for life-span increases in yeast, flies and Nvorms caused by caloric restriction or by natural antioxidants (e.g. resveratrol) (7, 8). Among the seven mammalian Sir2 homologs, SIRT1/Sir2alpha regulates skeletal muscle differentiation (9), represses damage-responsive Forkhead transcription factors (3, 4), negatively controls p53 to promote cell survival under stress (reviewed in (10)), and promotes fat mobilization in white adipocytes (11). Human SIRT2 is associated with microtubules in the cytoplasm and can deacetylate alpha-tubulin (12). Though the role of mitochrondrial SIRT3 is unknown (13, 14), variability of the human SIRT3 gene is associated with survivorship in the elderly (15).
Sir2 proteins catalyze a unique protein deacetylation reaction that requires the co-enzyme NAD+, a key intermediate in energy metabolism. In this reaction, nicotinamide (vitamin B3) is liberated from NAD+ and the acetyl-group of substrate is transferred to cleaved NAD+, generating a novel metabolite O-acetyl-ADP ribose, OAADPr (16-20). Although genetic studies have linked Sir2 to diverse phenotypes, few reports have investigated the biological function(s) of OAADPr and its possible connection with the observed Sir2-dependent biology. It has been suggested that OAADPr might be a substrate for other linked enzymatic processes, an allosteric regulator, or a second messenger. The first report of bio-activity came from the observation that OAADPr injected into starfish oocytes or blastomeres caused a block/delay in maturation and cell division, respectively (21). Enzymes capable of metabolizing OAADPr have been detected in several diverse cells (22). In vitro, select members of the ADP-ribose hydrolase (Nudix) family of enzymes (e.g., mNudT5 and yeast YSA1) are capable of efficient hydrolysis of OAADPr, while others like human Nudt9 are not (22). Interestingly, the long transient receptor potential channel 2 (TRPM2) has been shown to contain a C-terminal Nudix box (23). This Nudix motif is proposed to bind ADP-ribose, stimulating the Ca2+ channel activity of TRPM2 by initiating conformational changes leading to the opening of the channel pore (23-25). These findings prompted us to examine whether the TRPM2 ADP ribose gated Ca2+ channel could be activated/regulated by OAADPr.